Effects of Row Spacing and Planting Pattern on Photosynthesis, Chlorophyll Fluorescence, and Related Enzyme Activities of Maize Ear Leaf in Maize–Soybean Intercropping

Abstract

With the continuous improvement of the mechanization level and the development of new crop varieties, the optimal strip width for intercropping crops is important. In this study, field experiments were conducted to analyze the effects of different row spacings and planting patterns on photosynthesis, chlorophyll fluorescence, and the related enzyme activities of maize ear leaves in a maize–soybean intercropping system using two planting patterns (wide–narrow rows of 80–50 cm and uniform ridges of 65 cm) and two intercropping ratios (four rows of maize and four rows of soybean; six rows of maize and six rows of soybean). The results showed that the pattern of wide–narrow-row spacing significantly improved the photosynthetic capacity of maize compared with the uniform-ridge planting pattern, along with marked elevation in the values of stomatal conductance (Gs), the transpiration rate (Tr), and the photosynthetic rate (Pn). On the other hand, the values of photochemical quenching (qP), actual photochemical efficiency (φPSII), and maximum photochemical efficiency (Fv/Fm) also significantly increased, and the effect of D-M6S6 treatment was the most significant on these parameters. Similarly, the activities of phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase also increased significantly. Among different treatments, the yield under the D-M6S6 treatment was the highest. Therefore, based on the planting pattern of the wide–narrow-row spacing, the intercropping of six rows of maize and six rows of soybean is the better design in the semi-arid regions of western China.

1. Introduction

Maize (Zea mays L.) is one of the main crops in the northeast region of China. Reasonable plant-row spacing and planting patterns are the key factors for high-yield and efficient crop cultivation [1]. Under uniform-ridge conditions, the uniformity of the maize population is higher, and higher yields can be obtained [2]. However, the competition between plants is greater, and mutual shading between plants results in poor light transmittance [3]. Research showed that different row spacing arrangements of “20 + 20 + 40” cm increased average grain yields by 10% above the conventional 30 cm uniform spacing in three growing seasons (2009–2012) [4]. The pattern optimizes the canopy structure due to the marginal effect of maize and the significant improvement in the utilization of light energy, water, and fertilizer [5]. With the advancement of cultivation technology and the development of intensive agriculture, the yield of maize has reached a very high level in recent years, but accompanying problems, such as pesticide residues, fertilizer loss, and environmental pollution, have also become increasingly prominent [6]. As a “green revolution” strategy, intercropping can effectively improve land utilization and crop yield and also reduce environmental pollution [7]. Maize and soybean (Glycine max) intercropping is considered to be one of the preferred and effective intercropping systems [8]. Studies have shown that the maize–soybean intercropping model serves the functions of both maintaining fertilizer and improving the farmland ecosystem [9]. This system can effectively use the complementarity of the morphological and physiological features of the two crops, improve the efficiency of nutrient use, and ultimately increase crop yield and, consequently, improve the economic benefits [10,11]. Compared to maize and soybean monocultures, the yield of maize significantly increased with the four ratios: two rows of maize and two rows of soybean (2:2), four rows of maize and four rows of soybean (4:4), four rows of maize and two rows of soybean (4:2), and six rows of maize and six rows of soybean (6:6) [12].

In crops, dry matter accumulates through photosynthesis, and 90%–95% of these assimilates directly contribute to the crop yield [13]. Therefore, improving the maize photosynthetic performance is a basic approach to increasing the yield of maize [14]. Both gas exchange parameters and chlorophyll fluorescence parameters are important indicators of the efficiency of photosynthesis [15]. Leaves are not only the main photosynthetic organ of most land plants, but they also determine root growth and the corresponding acquisition of water and mineral nutrients [16]. The photosynthetic capacity of maize ear leaves affects its yield to a large extent, and the photosynthetic characteristics and physiological senescence indicators of its leaves can reflect the photosynthetic capacity of maize [17]. In the maize–soybean intercropping system, the inter-specific or intra-specific mutual shading caused by the planting structure directly changes the optical density between the maize and soybean canopy, enhances the light-trapping ability of maize, and makes its stomatal conductance (Gs), transpiration rate (Tr), and photosynthesis rate (Pn) increase [18]. Studies showed that in the intercropping system of four rows of maize and six rows of peanut, the photosynthesis rate significantly increased, and the light conditions were also improved [19].

The western area is a semi-arid region in Jilin Province, where there is long-term and continuous cultivation of maize, the soil productivity is reduced, and available nutrients become insufficient. We know that the intercropping system of maize and soybean has higher yield benefits. However, with the continuous increase in mechanization, the traditional maize–soybean intercropping model cannot adapt to the whole mechanized process of modern crop production. Therefore, the main objectives of this study were to evaluate the effects of different row spacings (four rows of maize and four rows of soybean; six rows of maize and six rows of soybean) and planting patterns (wide–narrow-row spacing and uniform ridges) on photosynthesis, chlorophyll fluorescence, and the related enzyme activities of maize ear leaf in a maize–soybean intercropping system and investigate the compound yield and land-use efficiency. The results will provide a theoretical basis for in-depth research on the mechanism of maize yield under the condition of wide–narrow rows.

2. Materials and Methods

2.1. Experimental Site

Field trials were conducted in 2018 and 2019 at the western research farm of the Faculty of Jilin Agricultural University (longitude 124°48′ E and latitude 45°08′ N). The average monthly rainfall and temperature within two years are shown in Figure 1. The soil of the test site was chernozem, the organic matter content in the 0–20 cm soil layer was 1.40%, the total nitrogen content was 2.133 g kg⁻¹, the total phosphorus content was 353.83 mg kg⁻¹, the contents of alkaline hydrolysis nitrogen, available phosphorus, and available potassium were 75.91 mg kg⁻¹, 16.31 mg kg⁻¹, and 130.24 mg kg⁻¹, respectively, and the soil pH value was 7.24.

2.2. Experimental Design

The tested maize hybrid, Hengdan 188 (128 days from emergence to maturity), was provided by Jilin Province Hengchang Agricultural Development Co., Ltd in Changchun, Jilin Province, China. Jinong 40 (127 days from emergence to maturity) was the soybean variety, which was obtained from the Agricultural College of Jilin Agricultural University. Four different treatments were designed with three replicates and 13,000 m² each, in line with mechanized harvesting. The four treatments included intercropping with 4 rows of maize and 4 rows of soybean in uniform-ridge mode (S-M4S4), 4 rows of maize and 4 rows of soybean in wide–narrow-row mode (D-M4S), 6 rows of maize and 6 rows of soybean in uniform-ridge mode (S-M6S6), and 6 rows of maize and 6 rows of soybean in wide–narrow-row mode (D-M6S6). The schematic figure of treatments is shown in Figure 2. The wide–narrow-row planting pattern (D) was adopted with a narrow row spacing of 50 cm and a wide row spacing of 80 cm. The uniform-ridge planting pattern (S) was adopted with an equal-row spacing of 65 cm. All treatments were repeated 3 times.

The sowing of maize and soybean was performed simultaneously on 28 April 2018 and 29 April 2019, respectively. The planting density of maize and soybean was 70,000 plants ha⁻¹ and 200,000 plants ha⁻¹, respectively. The amount of fertilizer applied to maize was 230 kg of N ha⁻¹,120 kg of P₂O₅ ha⁻¹, and 160 kg of K₂O ha⁻¹. Only 30% of total N was applied initially, with P₂O₅ and K₂O being the base fertilizers. Additionally, during the later stages of growth, the rest of the N fertilizer was applied as a top dressing. For the soybean crop, the applied amounts of P₂O₅ and K₂O were 60 and 25 kg ha⁻¹, respectively. The total amounts of these fertilizers were applied together as the base fertilizer. Maize and soybeans were harvested at the same time on 30 September 2018 and 28 September 2019, respectively.

2.3. Equipment and Methodology

Six maize plants with consistent growth in each plot and three repetitions were randomly evaluated, and a total of eighteen plants were evaluated for each treatment. Maize ear leaves were selected, and the LI-6400 photosynthesis system (LI-Cor, Lincoln, NE, USA) was used to measure the Pn, Tr, and Gs of ear leaves at 0 (July 25th), 10 (August 4th), 20 (August 14th), 30 (August 24th), 40 (September 3rd), and 50 (September 13th) days after the silking period of maize from 9:00 to 11:30 in the morning (sunny and cloudless). During the measurement, a fixed light source of 1200 μmol·m⁻²·s⁻¹ was used, the atmospheric CO₂ concentration was 380 ± 5 μmol·mol⁻¹, and the leaf chamber temperature was 25 °C.

The chlorophyll fluorescence was measured on the same leaf. A portable chlorophyll fluorescence apparatus (Mini-PAM, Walz, Germany) was used to measure the dark adaption maximum/minimum fluorescence (Fm/Fo) and the maximum/minimum fluorescence under light (Fm′/Fo′). Before measuring chlorophyll fluorescence, leaf samples were adapted to the dark for 20 min. Very low modulated light (<0.1 μmol (photon) m⁻² s⁻¹) was used to determine the minimum fluorescence yield in the dark-adapted state (Fo). The maximum fluorescence of the light-adapted state (Fm) and Fv/Fm were determined by a 3000 ms saturated light pulse. To determine the steady-state (Fs) and maximum fluorescence in the light-adapted state (Fm’), leaves were illuminated with actinic light (1800 μmol (photon) m⁻² s⁻¹). Using the formulas given below, the actual PSII photochemical efficiency (φPSII), maximum PSII quantum yield (Fv/Fm), and photochemical quenching (qP), together with non-photochemical quenching (NPQ), were determined [20,21].

Fv/Fm = (Fm − Fo/Fm)

(1)

qP = (F’m − F)/(F’m − Fo)

(2)

ΦPSII = (F’m − F)/F’m

(3)

NPQ = (Fm − F’m)/F’m

(4)

Then, the ear leaves were removed and put into an ice box to determine the following enzyme activities in the laboratory. The enzymes were extracted from 0.5 g of leaf tissue that was ground to a fine homogenate in 3 mL of 100 mM precooled Tris-HCl buffer (including 5% glycerol, 1% PVP, 1 mM EDTA, and 10 mM β-mercaptoethanol, pH 8.2) with a precooled mortar and pestle. The homogenate was centrifuged for 20 min at 15,000× g at 4 °C. The crude enzyme extract was stored at 4 °C before use.

The activity of phosphoenolpyruvate carboxykinase (PEPC) and Rubp carboxylase/oxygenase (RuBisCO) were measured according to the method described in Li and Li (1989), with slight modifications. The reaction solution of PEPC contained 0.1 mL of 100 mM Tris-HCl buffer (pH 9.2), 0.1 mL of 10 M MgCl₂, 0.1 mL of 10 mM NaHCO₃, 0.2 mL of 40 mM PEP (phosphoenolpyruvate), 0.3 mL of 1 mg ml⁻¹ NADH (pH 8.9), and 0.3 mL of malate dehydrogenase. The reaction solution was preheated for 10 min in a 28 °C water bath before the reaction was started by adding 0.1 mL of PEPC enzyme extraction solution. The reaction solution of RuBisCO contained 1 M Tris-HCl buffer (pH 8.0), 0.1 M MgCl₂, 1 mM EDTA, 50 mM ATP, 50 mM dithiothreitol (DTT), 2 mM NADH (3 mL of each above reagent), 0.1 mL of 200 mM NaHCO₃, 0.8 mL of ddH₂O, and 0.1 mL of 9 mM RuBP. The reaction solution was preheated for 10 min in a 30 °C water bath before the addition of 0.1 mL of a mixture of 3-phosphoglycerate kinase and 3-phosphoglyceraldehyde dehydrogenase. The measurement time was 1 min at 340 nm, and the results are expressed in μmol g⁻¹ h⁻¹.

SOD activity was determined by using nitro-blue tetrazolium (NBT) in the presence of riboflavin [22], POD activity was determined via the colorimetric method [23], CAT activity was determined via the ultraviolet absorption method [24], APX activity was measured by Wu et al.’s method [25], the O²⁻ level was detected by Wang et al.’s method [26], and MDA content was determined using thiobarbituric acid (TBA) [27]. In the same way, 0.5 g of fresh leaves was weighed and ground into a homogenate in acetone (10 mL), which was poured into a 10 mL centrifuge tube and centrifuged at 12,000× g for 20 min. Finally, the supernatant was collected, and the H₂O₂ content was determined according to the method of [28].

When the corn and soybean were physiologically mature, all of the rows of the plants were harvested from each plot; ten representative plants were selected to determine the number of ears and grains, and the number of pods and effective grains per pod of soybean were determined. After natural air drying, the 100-grain weights of corn and soybean were recorded, and the water content was measured with a special water tester. The maize and soybean yields of the community in both 2018 and 2019 were converted into 14% water content and then converted into yield per unit area.

2.4. Statistical Methods

Data are represented as mean ± standard error. One-way ANOVA was used to analyze data using SPSS 19.0. Each stage was analyzed individually, and Duncan’s test was used to determine differences across diverse treatments (p = 0.05).

3. Results

3.1. Gas Exchange Parameters

As the maize crop was reaching maturity after flowering, the Gs, Tr, and Pn of the ear leaves of maize cultivated in the uniform-ridge and wide–narrow-row patterns initially increased and then later decreased. However, there were significant differences in parameters between the two planting patterns (

Table 1. Effects of planting pattern and row spacing on the photosynthetic characteristics of maize ear leaves.

Index	Treatment	Days after Anthesis
		0d	10d	20d	30d	40d	50d
Pn	D-M4S4	30.8 ± 1.59 b	32.9 ± 3.20 b	36.5 ± 2.13 b	35.0 ± 2.79 a	29.6 ± 2.22 a	24.2 ± 2.32 a
	D-M6S6	34.0 ± 2.91 a	36.1 ± 2.59 a	40.7 ± 1.75 a	36.1 ± 1.92 a	32.1 ± 2.31 a	26.3 ± 1.77 a
	S-M4S4	21.8 ± 1.82 c	28.1 ± 1.92 c	30.4 ± 2.73 c	28.2 ± 1.54 b	20.9 ± 1.72 b	17.4 ± 1.76 b
	S-M6S6	22.8 ± 2.33 c	28.8 ± 2.92 c	29.4 ± 2.96 c	24.7 ± 2.79 c	20.6 ± 2.36 b	18.9 ± 2.17 b
Tr	D-M4S4	5.56 ± 0.60 a	5.99 ± 0.50 a	6.21 ± 0.72 a	5.52 ± 0.41 b	5.06 ± 0.69 b	4.31 ± 0.52 a
	D-M6S6	5.71 ± 0.35 a	5.90 ± 0.50 a	6.54 ± 0.59 a	6.16 ± 0.42 a	5.87 ± 0.47 a	4.49 ± 0.63 a
	S-M4S4	4.47 ± 0.50 b	5.14 ± 0.31 b	5.19 ± 0.40 b	4.5 ± 0.51 c	4.36 ± 0.46 c	3.51 ± 0.45 b
	S-M6S6	4.42 ± 0.38 b	5.45 ± 0.46 b	4.84 ± 0.51 c	4.27 ± 0.59 c	4.07 ± 0.49 d	3.13 ± 0.57 c
Gs	D-M4S4	0.51 ± 0.04 b	0.55 ± 0.03 b	0.64 ± 0.03 a	0.58 ± 0.04 a	0.51 ± 0.05 b	0.36 ± 0.04 a
	D-M6S6	0.59 ± 0.05 a	0.64 ± 0.04 a	0.68 ± 0.03 a	0.61 ± 0.02 a	0.55 ± 0.03 a	0.40 ± 0.04 a
	S-M4S4	0.38 ± 0.02 c	0.55 ± 0.02 b	0.57 ± 0.04 b	0.45 ± 0.03 b	0.42 ± 0.05 c	0.19 ± 0.03 b
	S-M6S6	0.39 ± 0.03 c	0.51 ± 0.02 b	0.52 ± 0.03 b	0.50 ± 0.03 b	0.43 ± 0.03 c	0.24 ± 0.06 b

Note: Different lowercase letters in the table indicate significant differences among treatments (p < 0.05) at the same time: 0 days after flowering is July 25; 10 days after flowering is August 4; 20 days after flowering is August 14; 30 days after flowering is August 24; 40 days after flowering is September 3; and 50 days after flowering is September 13 (the same applies to the following tables).

). The average Pn of the leaf at the ear position after anthesis was 29.25% and 42.9% higher in the wide–narrow-row pattern than in that of the uniform ridge for 4:4 and 6:6 intercropping ratios, respectively. Similarly, Tr was higher in the wide–narrow-row pattern than in that of the uniform ridge, which was 27.05% higher with the 4:4 intercropping ratio and 32.3% higher with the 6:6 intercropping ratio. The Gs value was increased by 23.21% with the 4:4 intercropping ratio and by 35.42% with the 6:6 intercropping ratio in the wide–narrow-row pattern compared with uniform ridges. In the same planting pattern, the values of these three parameters were higher with the 6:6 ratio of maize and soybean intercropping compared to the 4:4 intercropping ratio. However, the differences were not statistically significant.

3.2. Chlorophyll Fluorescence Parameters

Over time, the values of Fv/Fm, φPSII, and qP of the ear leaves of maize cultivated in the two planting patterns initially showed an increasing trend and then decreased, representing a single-peak curve. The NPQ values showed an opposite trend, representing a “V”-shaped curve. When the same intercropping ratio was maintained, following anthesis, the values of Fv/Fm, φPSII, and qP of the ear leaves of maize cultivated under wide–narrow-row planting pattern were higher relative to those of uniform-ridge maize and soybean intercropping (

Table 2. Effects of planting pattern and row spacing on chlorophyll fluorescence characteristics of maize.

Index	Treatment	Days after Anthesis
		0d	10d	20d	30d	40d	50d
Fv/Fm	D-M4S4	0.80 ± 0.089 a	0.81 ± 0.088 a	0.83 ± 0.083 a	0.77 ± 0.085 a	0.73 ± 0.086 a	0.69 ± 0.084 a
	D-M6S6	0.81 ± 0.124 a	0.83 ± 0.089 a	0.84 ± 0.085 a	0.76 ± 0.084 a	0.74 ± 0.088 a	0.72 ± 0.090 a
	S-M4S4	0.69 ± 0.193 b	0.74 ± 0.083 b	0.79 ± 0.092 b	0.69 ± 0.091 b	0.61 ± 0.087 b	0.51 ± 0.092 b
	S-M6S6	0.67 ± 0.224 b	0.72 ± 0.082 b	0.7 ± 0.079 b	0.64 ± 0.087 b	0.61 ± 0.091 b	0.52 ± 0.083 b
ΦPSⅡ	D-M4S4	0.57 ± 0.04 a	0.59 ± 0.04 b	0.61 ± 0.05 a	0.63 ± 0.03 a	0.53 ± 0.03 b	0.41 ± 0.02 b
	D-M6S6	0.59 ± 0.03 a	0.64 ± 0.03 a	0.64 ± 0.04 a	0.66 ± 0.02 a	0.58 ± 0.02 a	0.45 ± 0.03 a
	S-M4S4	0.45 ± 0.03 b	0.52 ± 0.04 c	0.58 ± 0.04 b	0.57 ± 0.04 b	0.42 ± 0.04 c	0.40 ± 0.03 c
	S-M6S6	0.44 ± 0.02 b	0.55 ± 0.03 c	0.55 ± 0.03 b	0.53 ± 0.05 b	0.42 ± 0.03 c	0.41 ± 0.02 b
qP	D-M4S4	0.52 ± 0.03 a	0.59 ± 0.04 a	0.65 ± 0.04 ab	0.60 ± 0.04 a	0.57 ± 0.04 b	0.49 ± 0.04 a
	D-M6S6	0.55 ± 0.03 a	0.55 ± 0.05 b	0.69 ± 0.04 a	0.65 ± 0.04 a	0.61 ± 0.05 a	0.52 ± 0.05 a
	S-M4S4	0.51 ± 0.04 b	0.52 ± 0.04 c	0.51 ± 0.03 c	0.51 ± 0.03 b	0.47 ± 0.05 c	0.38 ± 0.03 b
	S-M6S6	0.50 ± 0.05 b	0.56 ± 0.05 ab	0.62 ± 0.03 b	0.60 ± 0.05 a	0.46 ± 0.05 c	0.36 ± 0.05 b
NPQ	D-M4S4	0.54 ± 0.04 c	0.50 ± 0.03 b	0.47 ± 0.03 c	0.51 ± 0.03 b	0.61 ± 0.03 c	0.65 ± 0.02 b
	D-M6S6	0.55 ± 0.04 c	0.47 ± 0.04 c	0.41 ± 0.04 c	0.58 ± 0.03 b	0.65 ± 0.03 b	0.66 ± 0.03 b
	S-M4S4	0.71 ± 0.05 a	0.67 ± 0.05 a	0.64 ± 0.04 a	0.69 ± 0.05 a	0.74 ± 0.03 a	0.78 ± 0.05 a
	S-M6S6	0.60 ± 0.04 b	0.57 ± 0.02 b	0.52 ± 0.03 b	0.68 ± 0.03 a	0.73 ± 0.05 a	0.78 ± 0.02 a

Note: Different lowercase letters in the table indicate significant differences between the treatment groups at the same time.

). For the 4:4 intercropping ratio, the average values of Fv/Fm, φPSII, and qP on different days were 12.96%, 12.51%, and 15.21% higher, respectively, in maize cultivated in the wide–narrow-row planting pattern (D-M4S4) than in that cultivated in the uniform-ridge pattern (S-M4S4). Similarly, for the 6:6 intercropping ratio, the average values of Fv/Fm, φPSII, and qP on different days were 14.72%, 18.54%, and 13.16% higher, respectively, in maize cultivated in the wide–narrow-row planting pattern (D-M6S6) than in that cultivated with uniform ridges (S-M6S6). However, the average NPQ values on different days were 22.46% and 14.43% higher in the uniform-ridge pattern than in the wide–narrow-row planting pattern with the 4:4 and 6:6 intercropping ratios, respectively. When the same planting pattern was maintained, the average values of various chlorophyll fluorescence parameters determined after anthesis with the 6:6 intercropping ratio increased relative to the 4:4 intercropping pattern. However, the differences in these values between the two intercropping ratios were not significant.

3.3. Activities of Key Enzymes Involved in Photosynthesis of Leaf at The Ear Position

The activities of PEPC and RuBisCO determined in the ear leaves of maize initially increased and then decreased during the growth stage (

Table 3. Effect of planting pattern and row spacing on the activity of key enzymes in maize ear leaves.

Index	Treatment	Days after Anthesis
		0d	10d	20d	30d	40d	50d
PEP	D-M4S4	228 ± 11.5 a	255.9 ± 10.2 b	272 ± 10.6 a	254.6 ± 10.8 b	239.5 ± 14.1 a	208.4 ± 12.6 a
	D-M6S6	229.5 ± 11.1 a	270.5 ± 9.7 a	284.2 ± 12.0 a	279.7 ± 10.4 a	254.9 ± 12.4 a	211.3 ± 13.3 a
	S-M4S4	198.5 ± 11.0 b	213.4 ± 11.1 c	220.5 ± 11.5 c	200.9 ± 11.8 c	175.7 ± 10.2 c	150.8 ± 12.7 c
	S-M6S6	205.4 ± 14.0 b	232.1 ± 10.3 c	235.6 ± 11.1 b	219.2 ± 10.8 c	195 ± 10.6 b	180.0 ± 11.1 b
RUBP	D-M4S4	114 ± 11.4 a	122 ± 12.1 a	124.9 ± 8.1 a	107.4 ± 6.4 a	95.8 ± 6.3 a	85.0 ± 8.9 a
	D-M6S6	110.4 ± 12.8 a	130 ± 12.2 a	125.2 ± 9.7 a	113.5 ± 9.4 a	95.6 ± 9.6 a	83.8 ± 9.5 a
	S-M4S4	97.4 ± 12.3 b	105.4 ± 11.3 b	95.5 ± 6.9 b	91.6 ± 6.9 b	85.7 ± 7.2 b	74.5 ± 9.4 b
	S-M6S6	97.0 ± 10.9 b	104.9 ± 8.2 b	96.4 ± 9.5 b	89.0 ± 8.7 b	85.1 ± 7.3 b	76.6 ± 10.6 b

Note: Different lowercase letters in the table indicate significant differences between the treatment groups at the same time.

). When cultivated with the same intercropping ratio, after anthesis, the average activity of PEPC in the maize grown in the wide–narrow-row pattern was 25.84% (in the 4:4 intercropping ratio) and 20.7% (in the 6:6 intercropping ratio) higher compared with the uniform-ridge pattern. Similarly, the activity of RuBisCO was increased by 18.0% (in the 4:4 intercropping ratio) and 19.98% (in the 6:6 intercropping ratio) in the wide–narrow-row pattern compared to uniform ridges. In the same planting pattern, the average PEPC and RuBisCO activities after anthesis in the leaf at the ear position in the 6:6 intercropping pattern were higher relative to the 4:4 intercropping pattern. However, the difference in the activities of these enzymes was not significant.

3.4. Antioxidant Enzyme Activity of Ear Leaves

The antioxidant enzyme activities in maize ear-leaf samples are shown in

Table 4. Effect of planting pattern and row spacing on the antioxidant enzyme activity of maize ear leaves.

Index	Treatment	Days after Anthesis
		0d	10d	20d	30d	40d	50d
SOD	D-M4S4	350.1 ± 6.1 b	343.1 ± 6.6 b	332.8 ± 5.3 b	299.4 ± 8.7 b	272.5 ± 5.1 b	253.4 ± 6.7 b
	D-M6S6	389.9 ± 7.6 a	387.8 ± 5.6 a	368.3 ± 5.3 a	342.1 ± 3.9 a	302.6 ± 4.1 a	284.8 ± 6.1 a
	S-M4S4	316.3 ± 5.2 c	323.8 ± 4.5 c	317.5 ± 5.9 b	305.2 ± 4.0 b	254.6 ± 5.4 c	225.5 ± 4.3 c
	S-M6S6	295.4 ± 5.1 d	293.6 ± 5.6 d	276.8 ± 5.7 c	264.6 ± 6.0 c	241.1 ± 6.6 c	193.5 ± 4.2 d
POD	D-M4S4	76.2 ± 2.4 a	90.7 ± 2.9 a	99.9 ± 2.5 b	98.2 ± 3.4 b	79.7 ± 2.5 b	70.7 ± 2.1 a
	D-M6S6	75.4 ± 2.4 a	95.5 ± 2.1 a	107.4 ± 2.8 a	110.8 ± 2.1 a	92.7 ± 2.1 a	71.5 ± 2.5 a
	S-M4S4	65.4 ± 2.1 b	81.8 ± 4.4 b	90.5 ± 2.8 b	96.1 ± 3.4 b	79.9 ± 2.6 b	61.8 ± 3.7 b
	S-M6S6	62.6 ± 3.4 b	69.6 ± 3.3 c	80.1 ± 2.6 c	83.4 ± 3.1 c	64.5 ± 2.7 c	56.7 ± 1.8 c
CAT	D-M4S4	236.7 ± 3.9 b	230.2 ± 3.8 c	212.7 ± 2.6 b	188.4 ± 3.9 c	184.1 ± 2.6 a	134.1 ± 2.3 b
	D-M6S6	270.8 ± 2.8 a	274.7 ± 4.1 a	260.1 ± 4.4 a	234.8 ± 2.4 a	196.8 ± 2.1 a	161.6 ± 3.5 a
	S-M4S4	241.5 ± 2.9 b	243.5 ± 2.7 b	244.9 ± 3.4 a	204.2 ± 3.1 b	176.6 ± 2.2 b	145.5 ± 2.6 b
	S-M6S6	218.2 ± 1.9 c	232.3 ± 4.2 c	213.5 ± 3.7 b	198.6 ± 5.5 c	144.8 ± 2.1 c	127.1 ± 3.0 c
APX	D-M4S4	8.2 ± 0.3 a	7.6 ± 0.3 a	6.4 ± 0.4 a	5.2 ± 0.4 a	4.1 ± 0.3 a	3.5 ± 0.3 a
	D-M6S6	7.7 ± 0.4 a	7.4 ± 0.2 a	6.4 ± 0.4 a	5.3 ± 0.3 a	4.3 ± 0.4 a	3.5 ± 0.3 a
	S-M4S4	6.5 ± 0.4 b	6.1 ± 0.3 b	5.3 ± 0.3 b	4.2 ± 0.3 b	3.9 ± 0.3 b	2.6 ± 0.3 b
	S-M6S6	5.5 ± 0.2 b	5.4 ± 0.3 c	4.0 ± 0.3 c	3.6 ± 0.3 c	2.7 ± 0.2 c	2.1 ± 0.3 c

Note: Different lowercase letters in the table indicate significant differences between the treatment groups at the same time.

. It was found that the SOD and POD activities of ear leaves slightly increased after anthesis and then gradually decreased, but the CAT and APX activities continued to decrease. The SOD activity of maize leaves under each treatment was higher during the early stage, the POD activity of maize leaves in each treatment was higher during the middle stage, and these enzyme activities were the highest in the plants under the D-M6S6 treatment. In 2018, with a 4:4 intercropping ratio, the SOD, POD, CAT, and APX values of corn planted in wide and narrow rows were 5.86%, 7.74%, 12.5%, and 18.28% higher than those of corn planted in uniform ridges. With an intercropping ratio of 6:6, the SOD, POD, CAT, and APX values of the wide–narrow-row pattern were 24.6%, 24.6%, 18.9%, and 32.65% higher than those of the uniform-ridge pattern, respectively. In the wide–narrow-row pattern, the activity of the samples under the D-M6S6 treatment was significantly greater than that of samples under the D-M4S4 treatment. In the uniform-ridge pattern, the enzyme activity of maize under the S-M4S4 treatment was significantly greater than that of the S-M6S6-treated ones.

3.5. Maize Grain Yield

For the same ratio of intercropping, the average yields of maize and soybean strips in the wide–narrow-row maize–soybean intercropping planting pattern in 2018 and 2019 showed a marked increase relative to the uniform-ridge maize–soybean intercropping planting pattern (

Table 5. Comparison of yield and LER between strip intercropping and single cropping of maize and soybean.

Treatment	Yield of Maize Strip (kg ha−1)	Yield of Soybean Strip (kg ha−1)	Composite Yield (kg ha−1)			LER	Composite Economic Value (¥ ha−1)
			Maize	Soybean	Compound Yield		Maize	Soybean	Compound Value
D-M4S4	13,333.3 ab	2366.4 a	6666.6 a	1183.2 a	7849.8 a	1.16 ab	18,533.22 a	7099.2 a	25,632.42 a
D-M6S6	13628.8 a	2453.2 a	6814.4 a	1226.6 a	8041.0 a	1.20 a	189,44.03 a	7359.6 a	26,303.63 a
S-M4S4	12685.2 c	2034.3 c	6342.6 b	1017.2 b	7359.8 b	1.13 b	17,632.43 b	6102.9 b	23,735.33 b
S-M6S6	12764.2 bc	2201.4 b	6382.1 b	1100.7 b	7482.8 b	1.17 a	17,742.29 b	6604.2 b	24,346.49 b

Note: Different lowercase letters in the table indicate significant differences between the treatment groups at the same time. The average price of maize is CNY 2.78 ha⁻¹, and soybean is CNY 6 ha⁻¹.

). When grown in the same planting pattern, the maize yield of maize–soybean intercropping was higher compared to the monocropping pattern, and soybean production showed the opposite trend. The compound yield of the corn–soybean intercropping belt showed the overall trend of D-M6S6 > D-M4S4 > S-M6S6 > S-M4S4. The compound yield of D-M6S6 treatment was increased by 7.46% compared to that of S-M6S6, and the compound yield of D-M4S4 treatment was increased by 6.66% compared to that of S-M4S4. D-M6S6 had the highest LER value, 1.20, and the compound economic value was CNY 26,303.63 ha⁻¹.

4. Discussion

Photosynthesis is an important process for the synthesis of assimilation products [29]. Therefore, it can be used to improve plant growth and crop yield by increasing photosynthesis [30,31]. Planting a combination of C₃ and C₄ crops can change the ventilation and give full play to their spatially complementary advantages [32]. The plant height and photosynthetic light saturation level for photosynthesis in C₄ crops are higher than those of C₃ crops [33]. Previous studies have shown that high-position crops in the intercropping population have the advantage of light competition, which improves the photosynthetic rate and the utilization rate of light energy [34,35,36]. Our results also showed that intercropping changed the Pn, Tr, and Gs values of the maize ear leaf after anthesis, and all of the values on different days were represented by a single-peak curve, reaching their peak values after 10–20 days of anthesis and then decreasing.

The row ratio configuration affected the competitive dynamics between intercropped species by changing the canopy structure and light conditions of crops [37]. In our study, the wide–narrow-row planting pattern (80–50 cm) had relatively higher photosynthetic parameters compared to uniform-ridge planting (60 cm), because the change in ridge spacing not only changed the light energy distribution of interspecific populations but also changed the canopy microenvironment of populations within the same species. In a comparison of different strip widths, the average Pn, Tr, and Gs of D-M6S6 after anthesis were significantly higher than those of D-M4S4. This suggests that the intercropping ratio of six rows of maize and six rows of soybean with wide–narrow-row spacing efficiently utilized available resources.

Chlorophyll fluorescence acts as an internal probe to estimate the relationship between plant photosynthesis and the environment, which is important for determining the efficiency of light energy absorption, distribution, dissipation, and transmission during the process of leaf photosynthesis [38]. φPSII, Fv/Fm, NPQ, and qP are frequently used parameters to estimate the photosynthetic efficiency of plants [39]. Studies have shown that chlorophyll fluorescence is a better choice to measure if plants are affected by the environment and represents a simple and non-invasive method to analyze the effect of PSII on the efficiency of photosynthesis, as well as to understand how PSII responds to changes in the environment and growth conditions [40].

Fv/Fm represents the maximum photochemical quantum yield of PSII and is an important indicator of the photosynthetic performance of a plant. The larger the Fv/Fm value, the greater the light energy utilization potential of the plant [41]. In this study, the chlorophyll fluorescence parameter values Fv/Fm, φPSII, and qP of maize cultivated in the wide–narrow-row intercropping pattern significantly increased relative to the uniform-ridge intercropping pattern. Similarly, these parameters were higher in the 6:6 intercropping ratio than those observed in the 4:4 intercropping. The lower efficiency of the PSII reaction center of plants in the maize–soybean intercropping pattern under uniform-ridge conditions may be due to the dissipation of a large amount of absorbed light energy in the form of chlorophyll fluorescence, which in turn reduces the electron transfer through PSII [42]. Our study also showed that the NPQ value with the wide–narrow-row planting pattern was lower than that with the uniform-ridge pattern. NPQ is an indicator of the ability of plants to dissipate excess light energy as heat. In maize plants grown in the wide–narrow-row planting pattern, the NPQ value was lower, suggesting that in this planting pattern, the loss of energy in the form of heat at the PSII reaction center of maize is relatively low, more of the absorbed energy is used for photochemical reactions, and the light energy utilization is high [43].

PEPC and RuBisCO are the key enzymes for photosynthetic carbon assimilation in C₄ plants such as maize, and they also determine the degree of photosynthesis. Among these two enzymes, RuBisCO has dual functions of carboxylation and oxygenation, which can affect the efficiency of photosynthesis by regulating both photosynthesis and photorespiration. This enzyme has a significant impact on the net photosynthetic rate [44]. However, the catalytic efficiency of this enzyme is low, which is one of the important contributing factors to the limited photosynthetic efficiency [45]. PEPC plays a vital role in the fixation of primary CO₂ in C₄ plant leaves. Studies have shown that the introduction of PEPC into rice, wheat, and other C₃ crops significantly increases their rate of photosynthesis [46]. According to our findings, the enzyme activity of PEPC varied differentially in different planting patterns after flowering. The PEPC enzyme activity in the wide–narrow-row maize–soybean intercropping planting pattern was significantly higher than that in the uniform-ridge planting pattern. This increase was especially more evident during the first 20 days after flowering. After anthesis, the activities of PEPC and RuBisCO greatly increased, and the photosynthetic performance of leaves was also enhanced. This improvement was manifested in the increased production of photosynthetic products. The increased synthesis, transportation, and accumulation of photosynthetic assimilates ultimately increase the yield in maize [47].

In the late stages of maize growth, the leaves slowly begin to senesce [48,49]. In the anti-aging process of plants, antioxidant enzymes, including SOD, POD, CAT, APX, etc., play an important role and are responsible for maintaining the balance of active oxygen in the cell and protecting the cell membrane structure [50,51]. Active oxygen metabolism is an important metabolic process in the process of plant senescence, and accelerating this metabolism can alleviate the process of plant leaf senescence [52]. Our study found that the wide–narrow-row pattern significantly improved the SOD, POD, CAT, and APX activities of maize leaves at the flowering and grain stages and decreased the MDA content. Changing the row spacing and plant pattern made it possible to slow the aging of the crops. Green leaves take longer, and more dry matter can be transferred to the grain for high yields.

Crop yield is closely related to the accumulation of photosynthetic products. When the available light is insufficient, the net photosynthesis rate of the crop decreases, dry matter accumulation decreases, and subsequently, the yield decreases. A reasonable intercropping model is a useful way to effectively use the available resources, such as light, and take advantage of intercropping to increase the crop yield [53]. Intercropping can effectively increase the yield [54] and also improve land resource utilization [55]. However, soybean plants were affected by the shading caused by intercropping [56]. A wider distance between maize and soybean rows made it possible to achieve a higher grain yield for soybean [57]. In our study, compared to the uniform-ridge condition (65 cm), the average yields of maize and soybean under wide–narrow-row conditions (80 cm and 50 cm) significantly increased by 5.94% and 13.8%. Similarly, the compound yield obtained from the D-M6S6 treatment was the highest. LER was always used to determine the relationship between component crops [58].

5. Conclusions

The row spacing and planting pattern affected the population characteristics of maize–soybean intercropping. Compared with the traditional 65 cm uniform ridge, the plant pattern with wide–narrow-row spacing (80 cm and 50 cm) improved the photosynthetic capacity of maize, the values of various chlorophyll parameters, and the key enzyme activities of the ear leaves of maize. In addition, the wide–narrow-row intercropping pattern remarkably improved the compound yield of the intercropping population.

With the continuous increase in mechanization, the traditional maize–soybean intercropping model cannot adapt to the whole mechanized process of modern crop production in China. Therefore, in actual production, only a combination of agricultural machinery and agronomy can achieve a high and stable yield and high efficiency. According to our results, the intercropping mode with six rows of corn and six rows of soybean in the plant pattern with wide–narrow-row spacing is more effective in improving the light energy utilization rate of maize and thereby increasing the yield.